First evidence of ranunculids in Early Cretaceous tropics

Early Cretaceous floras containing angiosperms were described from several geographic areas, nearly from the Arctic to the Antarctic, and are crucial to understand their evolution and radiation. However, most of these records come from northern mid-latitudes whereas those of lower paleolatitude areas, such as the Crato Fossil Lagerstätte in NE Brazil, are less studied. Here, we describe from this region of northern Gondwanan origin, two fossil-species of eudicots belonging to a new extinct genus Santaniella gen. nov. Together with several vegetative axes and leaves, anatomically well-preserved fruits with seeds and persistent perianth-like organs allowed us to reconstruct its potential affinities with ranunculids, and presumably Ranunculaceae. Previous records putatively assigned to Ranunculales are all from mid-latitudes, and their first unequivocal occurrence in a low-latitude area supports further the hypothesis of a widespread radiation of the earliest diverging eudicot lineage by this early age.

Details of specimens. Geological setting and age. The study specimens are from the Crato limestones termed Pedra Cariri within a lithostratigraphic unit known as the Crato Formation, belonging to the Santana Group in the Jurassic-Cretaceous Araripe Basin (NE Brazil) 26 . They have been gathered at open cast pits close to the town of Nova Olinda (Ceará) by local workers who excavate the laminated lacustrine carbonate rock as cut and ornamental stone. Recent dating of other Santana Group formations assumed an early Aptian or even late Barremian age for the Crato Formation 27,28 . However, this age is not consensual since former estimations suggested a late Aptian/early Albian age of this geologic unit ( 29 and references therein). Due to the diversity, preservation and abundance of biota, as well as its paleogeographic and temporal context, the Crato limestones contain one of the most important Early Cretaceous fossil lagerstätten in Gondwana 30 .
Associated plant taxa. In the Crato Formation flora, the numbers of taxa range from more than 40 based on macrofossils and more than 150 based on palynomorphs of spore and seed-bearing plants, and not all were formally described yet 30,31 . In addition, the angiosperm component of the Crato Formation flora is diverse both taxonomically and ecologically 32 , and includes several fossil-taxa with clear affinities with early-diverging extant lineages (ANA grade 33,34 ) or mesangiosperms (e.g., magnoliids [35][36][37][38] , monocots 32,39 ). Several early eudicot taxa were reported earlier but none had been described yet 30  Generic diagnosis. Angiosperm with herbaceous to suffruticose growth habit. Thin elongated axes with leaves ranging from sessile and simple distally to increasingly longer, larger, and more divided and lobed towards the base. Proximal leaves pedately compound with typical bifurcation into two (type II) or more leaflets (type III). Leaf and leaflets unlobed to lobed with untoothed margin. Petiole (and petiolules) with marginal attachment. Primary venation pinnate and secondary craspedodromous, branching dichotomously and merging into a perimarginal vein. Anastomoses rare. Fruit a solitary and terminal follicetum derived from an apocarpous, superior gynoecium with 2 to up to at least 11 multiovulate plicate carpels developing into stipitate, reflexed and pendulous follicles, born on a short, flattened to slightly concave receptacle typically surrounded by persistent perianth-like organs. Fruitlet comprises at least up to 9 seeds arranged in a row along a median longitudinal placenta on the abaxial side.
Etymology. The genus name is derived from the lithostratigraphic unit known as the Santana Group where the studied specimens come from. showing several leaves and a terminal aggregate fruit, with 5-6 stipitate multi-seeded follicles (white arrows point to leaf attachment, gray arrow to a putative opposite leaf, and black arrows to pair of distal most leaves); (B) close-up on follicles attachment to the receptacle, surrounded by 3 outer reflexed (white arrows) and several inner erect and laciniate persistent perianth-like organs (black arrow); (C-E) close-up on leaves showing a gradual acropetal reduction in organization and size from (E) to (C); (C, D) leaf type II. (E) leaf type III with four lobed leaflets (black arrows point to primary veins). Scale bars = 1 cm.     Description and remarks. The holotype (MB.Pb. 1998/691) consists of a leafy axis with a terminal fruit (Fig. 1).
The axis is about 230 mm long and 2.6 mm wide, and bears several leaves showing a gradual acropetal reduction in both structure and size (Fig. 1A, C-E). The lowest and most proximal leaf is pedately compound and of type III (Fig. 5), with four overlapping lobed and petiolulate leaflets (Fig. 1E), which is better preserved in the second paratype (see below, Fig. 2D). The petiole is 21 mm long and 1.1 mm wide, and the leaf blade is microphyllous, longer and wider than 40 mm. The second and more distal leaf fragments are poorly preserved and can be interpreted as two leaflets of a single leaf of type II, or a pair of leaves of the same type in which the bifurcation is not clearly visible ( . They are nanophylls, 24 mm long and 2.8 mm wide. L/W ratio is ca. 8:1 and the overall shape is unclear. We do not exclude that they are subtending bracts or a pair of bracteoles. The aggregate fruit comprises 5-6 stipitate and pendulous follicles. They are 23-30 mm long and 11-12 mm wide, with an 8-8.5 mm long stalk (Fig. 1A). The follicles are connected to a receptacle surrounded by two types of persistent perianth-like organs. The three outermost organs are obovate, with an entire margin and ca. 10 mm long and 6 mm wide (Fig. 1B). Their original organization and number are uncertain but some parts indicate that they were free and reflexed. In contrast, the innermost organs are erect and laciniate (Fig. 1B).
The first paratype (MB.Pb. 1997/1347) is also a leafy axis with a fruit ( Fig. 2A). The axis is about 137 mm long and 2.6 mm wide. The most proximal leaf is also pedately compound and of type II (Fig. 5), with two overlapping 3-lobed petiolulate leaflets (Fig. 2C). The petiole is 14 mm long and 1.6 mm wide, and the leaf blade is microphyllous and 36 mm long by ca. 40 mm wide. L/W ratio is about 1:1 and the overall leaf shape is ovate and symmetrical. The leaflets are microphyllous and 36 mm long and 22 mm wide. Their base is acute and decurrent, whereas their lobes are obtuse and rounded. The most distal leaf is simple nanophyll of type I (Figs. 2A, 5) and 18 mm long and 5.2 mm wide. L/W ratio is about 3:1 and the lamina shape is obovate. It is unclear, however, whether there was a second (sub)opposite proximal leaf, and we do not exclude that the distal one is a subtending bract, or one of two bracteoles ( Fig. 2A).
The fruit of the first paratype is an aggregate and comprises 10-11 follicles. They are 21-25 mm long and 10-11 mm wide, with a 6-9 mm long stalk ( Fig. 2A, B). They are also surrounded at the base by two types of persistent perianth-like organs, comprising 4 outer organs that are very similar to the distalmost leaf, and inner organs with a large base and a more or less laciniate apex (Fig. 2B).
The second paratype (LP UFC CRT 1559a) is a single, well-preserved complete pedately compound leaf of type III (Fig. 5) showing successive dichotomous bifurcations resulting into four lobed asymmetrical leaflets with untoothed margins (Fig. 2D). The complete leaf is reconstructed as being 70 mm long and ca. 50 mm wide. The petiole is about 17 mm long and 1.6 mm wide up to the first bifurcation, and the leaf is a microphyll with a 43 mm long and ca. 50 mm wide blade. L/W ratio is about 1:1 and the lamina shape is ovate and symmetrical. Lamina is chartaceous. The leaflets are sessile and microphyllous and 35-43 mm long and 13-18 mm wide. Their base is acute and decurrent, whereas their lobes are obtuse and rounded. Their blades are also odd-pinnatisect with three to five alternate and asymmetrical lobes, which are obovate and asymmetrical in the middle, and increase in size acropetally. The lobes are proximally decurrent onto the primary vein, but end abruptly distally.
The overall architecture of the leaf (Fig. 5) is derived from a palmate organization with a petiole running straight and then bifurcating. The primary venation of leaflets is pinnate with a single basal vein and without naked basal veins. Agrophic veins and interior secondaries are absent. The major secondaries are craspedodromous and branch dichotomously two to three times before merging into a perimarginal vein. Rare anastomoses occur. The venation of the lobes is mainly composed of secondary veins, except for the distal lobes of each leaflet where their pinnate primary vein ends. Secondary venation is bifurcating and rather thin compared to the primary vein (0.08-0.22 mm), the width ratio secondaries ∕ primary is typically 1:2. The secondaries are regularly spaced (0.17-0.27 mm) and they form uniform moderately acute angles (30-45°) to the primary vein. The attachment of secondaries to the midvein is long decurrent, with the major ones running several millimeters subparallel to the midvein. Intersecondaries and higher order venation are absent. Vein density ranges of 4.5-4.9 (mean of 4.6 mm −1 ).
The third paratype (MB.Pb. 1997/1562) is a fruit with seeds (Fig. 3). The structure of the fruit is very similar to those of the holotype and first paratype, with 6 stipitate follicles surrounded by several innermost erect laciniate and one outermost obovate (reflexed) persistent perianth-like organs. The follicles are 24-29 mm long Scientific Reports | (2022) 12:5040 | https://doi.org/10.1038/s41598-022-07920-y www.nature.com/scientificreports/ and 10-13 mm wide, with a 7-11 mm long stalk (Fig. 3A). The seeds are elliptical and bilaterally symmetrical, and 3-5 mm long and 2 mm wide (Fig. 3A, B). The seed coat comprises two distinct layers, with the outermost composed of elongated cells with straight to undulate walls around the micropyle but forming a jigsaw puzzleshaped surface over the chalazal region ( Fig. 3C-E). A smooth papillate or punctate wall ornamentation with a spongy appearance is also present on the outer layer, especially at the periphery of the seed. In contrast, the inner layer is composed of more or less isodiametric polygonal to irregular cells which form a reticulum, and contain spiral structures which nature remains unclear (Fig. 3D). Below the seed coat, there is no evidence of an embryo, suggesting that it is surrounded by an endosperm and that the seed is albuminous.
The following features were compiled from characters of all specimens of the protologue. The follicles have on average an estimated volume of ca. 1215 mm 3 and display numerous transverse veins that are 0.2-1 mm apart. Each follicle contains several seeds (ca. 5-9) with on average an estimated volume of ca. 5.8 mm 3 , and arranged in a row with the micropyle facing the median longitudinal abaxial placenta. Description and remarks. The holotype (MB.Pb. 2000/71) and only specimen is a leafy axis with a fruit (Fig. 4). The axis is about 125 mm long and 2.5 mm wide. The two proximal leaves are pedately compound and of type II (Fig. 5). Their petioles are about 6-7 mm long and 1-1.6 mm wide, the leaves are microphylls with ca. 23 mm long and 15 mm wide blade. The L/W ratio is about 1.5:1 and lamina shape is ovate. Lamina is chartaceous. The leaflets are nanophyllous and 16-23 mm long and 5-6 mm wide. They are sessile, lobed to unlobed with untoothed margins, and slightly asymmetrical in the middle of the blade. Their base is acute and cuneate (asymmetrical), and their apices acute and straight to convex. In contrast, the distal-most leaves are simple and of type I (Figs. 4A, 5). One is unlobed and at least 10 mm long and 2.3 mm wide. The L/W ratio is about 4:1. The second distal leaf seems to be bilobed, but it is not well-preserved. However, we do not exclude because of their similarities that the proximal leaves were (sub)opposite and the distal leaves were a pair of bracts or bracteoles.
The overall architecture of the leaf (Fig. 5) is derived from a palmate organization with a petiole running straight and then bifurcating. The primary venation of leaflets is pinnate with a single basal vein and without naked basal veins. Agrophic veins and interior secondaries are absent. The major secondaries are craspedodromous and branch dichotomously two to three times before merging into a perimarginal vein (Fig. 4B). Rare anastomoses occur. Secondary venation is bifurcating and rather thin compared to the primary vein (0.08-0.2 mm), the width ratio between secondaries and the primary vein is typically 1:2 in the distal part of the leaflet. The secondaries are regularly spaced (0.15-0.3 mm) and they form uniform moderately acute angles (30-45°) to the primary vein. The attachment of secondaries to the midvein is long decurrent, with the major ones running several millimeters subparallel to the midvein. Intersecondaries and higher order venation are absent. Vein density is 4.5 mm −1 .
The fruit of the holotype is an aggregate and comprises 5-6 follicles surrounded by one obovate, persistent perianth-like organ. The fruitlets are 21-25 mm long and 12 mm wide, with a 7-9 mm long stalk (Fig. 4). They have on average an estimated volume of ca. 1203 mm 3 and display numerous transverse veins that are 0.2-0.6 mm apart. The follicles contain at least 5 seeds which are elliptical, bilaterally symmetrical, and 3-5 mm long and 2 mm wide. The seed volumes have on average ca. 5.8 mm 3 . Their follicles, seeds and perianth-like organ are similar to those of Santaniella lobata. However, there is no evidence of innermost laciniate, persistent perianthlike organs surrounding the follicetum in Santaniella acuta.
Cladistic analyses and potential systematics affinities. Our first cladistic analysis (Fig. 6A) was performed using the matrix by Kvaček et al. 40 and results in five most parsimonious positions of Santaniella within basal eudicots, either as sister to Glaucideae (Glaucidium), or Hydrastideae (Hydrastis), or Glaucideae + Hydrastideae (successively basal-most lineages of Ranunculaceae), or sister to Nelumbo or Proteaceae + Platanus (basal eudicots). However, if Santaniella is sister to Glaucidium, it would imply changes in distichous phyllotaxis (CH22), marginal teeth (CH35) and carpel number (CH97), and as sister to Hydrastis changes in distichous phyllotaxis (CH22), marginal teeth (CH35) and ovule direction (CH115). Similarly, the Glaucideae + Hydrastideae position implies changes in marginal teeth (CH35), carpel number (CH97) and ovule direction (CH115). If sister to Nelumbo, it would imply changes in leaf dissection (CH34), ovule direction (CH115) and fruit dehiscence (CH126). Also, Nelumbonaceae differ from Santaniella by having large, and centrally peltate leaves, the perianth Scientific Reports | (2022) 12:5040 | https://doi.org/10.1038/s41598-022-07920-y www.nature.com/scientificreports/ is caducous and each carpel forms a 1-seeded nutlet embedded in an enlarged and obconic receptacle 41 . Similarly, the Proteaceae + Platanus position would imply changes in inflorescence structure (CH42), ovule direction (CH115) and fruit dehiscence (CH126). Proteaceae also differ from Santaniella by having a unicarpellate gynoecium 42 , and Platanaceae by their usually distinct simple, toothed and broadly palmately lobed leaves, and long-pedunculate inflorescence with globular heads, and 1-seeded fruits interpreted as densely hairy achenes or nutlets in a globose head 41 . Inside Ranunculales, one step less parsimonious positions were recovered as sister to Ranunculaceae as a whole, or as sister to the core of the family. Other ranunculid lineages are two to four steps less parsimonious (see below the second cladistic analysis). One step less parsimonious position outside of the basal eudicots either falls within magnoliids in Piperales and would imply changes in leaf dissection (CH34), inflorescence structure (CH42), carpel number (CH97) and fruit wall (CH124), or within monocots in Dioscoreaceae, but would also imply changes in leaf dissection (CH34), inflorescence structure (CH42), carpel number (CH97) and carpel fusion (CH107). Other positions within other magnoliids or monocots are often less parsimonious and, in magnoliids, fall in lineages which often differ from Santaniella by the absence of several dehiscent follicular fruitlets with many seeds 2,43 and, in the case of monocots, can be excluded by their typical linear leaves with parallelodromous or acrodromous to campylodromous venation, and sheathing bases 38 . Positions within the ANA grade are three to four steps less parsimonious, but in contrast with Santaniella, none of their extant lineages have plicate carpels and/or several ovules, which are synapomorphies of most mesangiosperms 44 . Our second cladistic analysis using the dataset of Wang et al. 45 , which is more centered on Ranunculales, results in the most parsimonious positions of Santaniella within Ranunculales, as sister to Berberidaceae + Ranunculaceae, Berberidaceae, or Ranunculaceae (basal and core lineages), but also among basal eudicots, as sister to Lardizabalac.
Circ. www.nature.com/scientificreports/ Platanus + Proteaceae (Fig. 6B). No changes in any characters are expected in all the most parsimonious positions. However, if Santaniella is within of crown group of Berberidaceae, it would imply changes in carpel number (CH39) and carpel form (CH40), because they fundamentally differ by their single and ascidiate carpel 43 . The most parsimonious positions within the Ranunculaceae are widespread in basal and core lineages (except Callianthemeae and the last-diverging lineages, i.e., Anemoneae and Ranunculeae). While positions inside Ranunculales (except Ranunculaceae) as sister to Menispermaceaeae or Papaveraceae would be one step less parsimonious, they would imply changes in fruit dehiscence (CH56), or in major venation (CH19), respectively. In addition, Menispermaceae also differ from Santaniella by having simple leaves, and the flowers gathered in inflorescences which develop fruits 1-seeded drupes 41 , and Papaveraceae by their syncarpous gynoecia with 2, rarely 3 or up to 25 carpels 43 . Two steps less parsimonious and it could be sister to Circaeasteraceae and Lardizabalaceae, and imply similar changes in major venation (CH19), and fruit dehiscence (CH56). Also, Circaeasteraceae differ from Santaniella by having toothed leaves, achene fruits and 1 to 3 carpels in Circaeaster and 5 to 9 carpels in Kingdonia, but with one ovule in each carpel 41,43 . In Lardizabalaceae, the flowers are gathered in inflorescences and form fleshy fruits 41 .

Menispermaceae
In summary, our cladistic analyses suggest that Santaniella belongs to basal eudicots, most likely to the Ranunculales or the stem lineage of Ranunculaceae. Indeed, the combination of Santaniella features, especially of compound leaves and a superior apocarpous gynoecia with several carpels producing many seeds, is more common in basal eudicots than in any other main lineages of angiosperms 46,47 . Also, most of traits displayed by Santaniella occur singularly or in various combinations in several extant members of Ranunculales (e.g., in Ranunculaceae), such as the presence of compound and lobed leaves 41 , craspedodromous secondary venation without intersecondary veins 48 , bracts (or cauline bract-like leaves) along the stem 41 , solitary and terminal flowers with short receptacles 41 , superior apocarpous gynoecium with plicate carpels with transverse veins and several ovules 43 , aggregate fruits with stalked follicular fruitlets 41 , persistent floral organs at fruiting stage 49 , and the range of size and shape of seeds 50 . Unfortunately, the nature of the persistent perianth-like organs in Santaniella remains unclear, and despite the above similarities the unique combination of its vegetative and reproductive traits does not fit in any extant taxon of Ranunculales.
Some taxa with ranunculidean affinities from Liaoning province, China (late Barremian to early late Aptian age) were reported by Leng and Friis 12 , Dilcher et al. 51 , and Sun et al. 13 . Hyrcantha decussata (Leng & Friis) Dilcher et al. shares with Santaniella terminal fruits with multiple ovules or young seeds, but differs by its syncarpous gynoecium with 2-4 carpels and decussate phyllotaxy of infructescences 12,51 . Leefructus mirus Sun et al. differs from Santaniella by its simple and trilobate leaves with toothed margins, and syncarpous gynoecium with 5 carpels 13 . Another eudicot from the Early Cretaceous of China, Gansufructus saligna B. Du., is also preserved at the fruiting stage and bears some resemblances to Santaniella by its persistent perianth, superior gynoecium, and multiple seeds. However, it differs in having simple leaves with poorly organized leaf venation, paniculate infructescences, and tetracarpellate syncarpous gynoecia 14 .
From the late Barremian of Spain, Iterophyllum lobatum Barral et al., is possibly also a member of Ranunculales and resembles Santaniella lobata by its lobed pinnatipartite leaves with pinnate primary and craspedodromous secondary venation. However, its leaves are always simple with a dentate margin 15 .
Charcoalified mesofossils with apocarpous gynoecia and superior ovaries containing several ovules with similar affinities to basal eudicots/Ranunculales were also described from the late Barremian to middle Albian of Portugal and the United States. However, all these fossils, e.g., Kajanthus lusitanicus Mendes et al. 17 , Paisia pantoporata Friis et al. 18 , and Kenilanthus marylandensis Friis et al. 24 , are only known from preanthetic or anthetic flowering material which can hardly be compared to the fruits of Santaniella. There is also Teixeira lusitanica von Balthazar et al. 16 but it is a unisexual male flower without any evidence of carpels and therefore cannot be compared to Santaniella.
From Aptian to middle Albian Potomac Group sediments in the United States, putative eudicots were described as Potomacapnos apeleutheron Jud & Hickey, which is also characterized by lobed leaflets and veins that ramify dichotomously and fuse into a perimarginal vein like in Santaniella, but differs by its glandular teeth and reticulate venation 22 . The pinnately lobed leaves of Fairlingtonia thyrsopteroides Jud are also quite similar to those of Santaniella lobata with their pinnate primary veins and a craspedodromous secondary venation branching into perimarginal veins, but they have small petioles, and lobes which decrease in size acropetally and end with a glandular papillate tooth 23 .
Another potential eudicot, Baderadea pinnatissecta Pessoa et al., was recently described from the Crato Formation flora 6 . It consists of a single leaf with a pinnatisect dissection similar to that of the leaflets of Santaniella lobata, but the leaf is simple and its lobes are distinctly linear. In addition, its potential affinities with eudicots are only supported by the leaf dissection and primary venation.
Overall importance of our findings. Nowadays, many Ranunculales occur in areas where they could potentially undergo a fossilization process 2 . However, most of them are typically herbaceous and, in contrast to forest canopy-forming trees or shrubs, are less likely to be preserved as fossils, and also usually produce and shed less vegetative and reproductive parts over their life time 52 . In addition, they can also occur in environments characterized mostly by erosional rather than depositional processes, and thus, are even less likely to be buried within sediments 52 .

Scientific Reports
| (2022) 12:5040 | https://doi.org/10.1038/s41598-022-07920-y www.nature.com/scientificreports/ Fortunately, the depositional lacustrine environment of the Crato Formation along with the microbial mats enables the remarkable preservation potential of various organisms 53 , including herbaceous and (sub)shrub-like plants less susceptible to be preserved in the fossil record 30,54,55 . Therefore, the Crato Formation flora allows to track the evolutionary history of plant lineages that are usually not or underrepresented in the fossil record, and is also remarkable by its diversity, especially of early-diverging and basal angiosperm lineages such as Nymphaeales and magnoliids, as well as monocots and eudicots 55 . It also demonstrates that all major flowering plant lineages were already diversified and lived in low latitudes under hot and (seasonally) dry conditions by the middle Early Cretaceous.
Earlier evidence of eudicots at low paleolatitudes in the Early Cretaceous comes from tricolpate pollen 56,57 and is now confirmed by the macrofossils described herein. These new Brazilian fossils are the most complete macrofossils of an early-diverging eudicot with both vegetative and reproductive structures strongly supporting affinities to ranunculids in Early Cretaceous tropics. With all other fossil records linked to Ranunculales from northern mid-latitudes, our results do not only support that the widespread radiation of this clade already started at this early age, but also that they were present in paleoequatorial tropical areas.

Methods
Fossil analysis. The material was studied using a Leica Wild M10 microscope equipped with a Leica DFC 425 camera. Macroscopic images were taken with Canon 250D camera. One specimen exhibits remaining permineralized tissues with cellular structures, especially of in-situ seeds, that were replaced by iron oxide. The complete fossil (MB. Pb. 1997/1562) was directly placed uncoated inside the chamber of a Zeiss EVO 50 Scanning Electron Microscope (SEM) (Carl Zeiss, Germany), and characteristics of the epidermis were observed under a low vacuum. SEM photographs were edited with Photoshop software.
Leaf architecture. The classes of leaf size and part of leaf architecture descriptions follow Ellis et al. 58 .
Leaf width inferences. The leaf width measurements of leaf types II (Fig. 2C) and III (Fig. 2D) were inferred based on the leaf reconstructions (Fig. 5), assuming that the leaflets were originally not overlapping. They were used to estimate the laminar size, L:W ratio, leaf shape, and medial symmetry of leaf in these specimens. Cladistic analysis. Two cladistic analyses using morphological characters of Santaniella were performed using first the data matrix of Kvaček et al. 40 with all major angiosperm lineages except core eudicots, and then using the data matrix of Wang et al. 45 which is more specific of Ranunculales. Phylogenetic analyses were conducted using Mesquite 3.61 60 , where alternative positions were explored and character states were reconstructed. Characters were unordered and equally weighted.